The isolated reactive site beta-hairpin loop of Bowman-Birk-type proteinase inhibitors has become a widely studied proteinomimetic because it retains the three-dimensional structure and much of the inhibitory potency of the corresponding region of the complete protein. Here we analyse the role of the P1' Ser residue which is highly conserved and intramolecularly hydrogen bonded in the complete proteins. A combined kinetic and structural analysis of variant proteinomimetic peptides demonstrates that the hydrogen-bond potential of the side-chain oxygen atom of the P1' Ser is not essential for the integrity of the reactive site loop and that it provides only a small contribution to the trypsin affinity and no apparent contribution to the stability against tryptic turnover. We conclude that the potential of the P1' side chain to engineer improved inhibition and selectivity for serine proteinases is best explored further in concert with the side chains of the P2 and P5' residues which may interact or compete for the same space. (C) 2003 Elsevier Inc. All rights reserved.